Protein prenyltransferases, such as protein geranylgeranyltransferase type I (GGTase-I) and Rab geranylgeranytransferase (RabGGTase), catalyze posttranslational modification of proteins, often involving the addition of isoprenoids (1-5). For example, protein farnesylation involves the addition of a C15 farnesyl group to proteins ending with the C-terminal CAAX motif (C is cysteine, A is an aliphatic amino acid and X is usually serine, methionine, glutamine, cysteine or alanine). Farnesylated proteins include Ras proteins, Rheb proteins, nuclear lamins and Hdj2.
Protein geranylgeranylation involves the addition of a longer isoprenoid, C20 geranylgeranyl group. Protein geranylgeranylation is critical for the function of a number of proteins such as RhoA, Rac and Rab.
Two different types of geranylgeranylation have been reported. Rho family proteins such as RhoA, Cdc42 and Rac as well as the γ-subunit of heterotrimeric G-proteins are geranylgeranylated at a cysteine within the CAAL motif (similar to the CAAX motif but the C-terminal amino acid is leucine or phenylalanine) at their C-termini. Rab proteins involved in protein transport across the secretory pathway and endocytosis pathway are also geranylgeranylated. These proteins usually end with CC (two cysteines) or CXC at the C-termini and both cysteines are geranylgeranylated.
Geranylgeranyl transferase type I (GGTase-I) catalyzes mono geranylgeranylation of proteins such as Rho, Rac and Cdc42. This enzyme is a heterodimer consisting of alpha and beta subunits (15). RabGGTase (or GGTase-II) catalyzes digeranylgeranylation of Rab proteins (16,17). This enzyme also contains alpha and beta subunits, but contains an additional subunit Rab Escort Protein (REP) (16,18). The REP subunit binds to the substrate Rab protein (19). The alpha and beta subunits share homology with corresponding subunits of GGTase-I.
Two prenyltransferases, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) are regarded as structurally similar enzymes. See Protein Lipidation (2001) The Enzymes vol. 21. (eds. Tamanoi, F. and Sigman D. S.) Academic Press. FTase consists of two subunits, alpha and beta. Its structure consists exclusively of alpha helices and the alpha subunit wraps around the beta subunit that forms a beta-beta barrel structure. GGTase I is also a heterodimer containing an alpha-subunit that is shared with FTase. Furthermore, the beta-subunit of GGTase I shares a significant similarity with the beta-subunit of FTase.
FTase catalyzes the transfer of a C15 farnesyl group from farnesyl pyrophosphate, an intermediate in cholesterol biosynthesis, to proteins such as Ras, Rheb, nuclear lamins, CENP-E, F and protein tyrosine phosphatases pRL1-3. See Tamanoi, F., Gau, C. L., Edamatsu, H., Jiang, C. and Kato-Stankiewicz, J. (2001) Protein farnesylation in mammalian cells, Cell Mol. Life Sci. 58, 1-14. These proteins end with the CaaX motif that is recognized by FTase. GGTase I catalyzes the transfer of a C20 geranylgeranyl group from geranylgeranyl pyrophosphate to proteins ending with the CaaL motif. Geranylgeranylated proteins include Rho, Rac, Cdc42 as well as gamma-subunit of heterotrimeric G-proteins.
Certain protein prenyltransferases have been implicated in cancer processes, including GGTase I, RabGGTase, and FTase. See Tamanoi, F., Gau, C. L., Edamatsu, H., Jiang, C. and Kato-Stankiewicz, J. (2001) Protein farnesylation in mammalian cells, Cell Mol. Life Sci. 58, 1-14; Carrico, D., Blaskovich, M. A., Bucher, C. J., Sebti, S. M., Hamilton, A. D. (2005) Design, synthesis, and evaluation of potent and selective benzoyleneurea-based inhibitors of protein geranylgeranyltransferase-I, Bioorg. Med. Chem. 13, 677-688; Peterson, Y. K., Kelly, P., Weinbaum, C. A., Casey, P. J. (2006) A Novel Protein Geranylgeranyltransferase-I Inhibitor with High Potency, Selectivity and Cellular Activity, J. Biol. Chem. 
Further studies also demonstrate the physiological significance of protein geranylgeranylation, for example, in cancer. Knockout mice specific for the beta-subunit of GGTase-I have been established (6). Characterization of GGTase-1-deficient cells showed proliferation inhibition and accumulation of p21CIP1/WAF1, pointing to the significance of GGTase-I in cell proliferation and cell cycle progression (6). GGTase-I deficiency reduced oncogenic K-ras-induced lung tumor formation in mice, pointing to the significance of inhibiting GGTase-I to block tumor formation (6). Recent studies also showed that a number of geranylgeranylated proteins play important roles in tumorigenesis and metastasis. In addition to RhoA and Cdc42 proteins, RalA protein was recently found to be activated downstream of Ras in most pancreatic cancer cells harboring oncogenic K-ras mutation (7). RalB plays critical roles in the survival pathway (8). RhoC is overexpressed in metastatic cancer and RhoC knockout mice exhibit defect in metastasis (9,10). Overexpression of Rab25 in breast and ovarian cancer cells has been reported, and this mutation is a determinant for aggressiveness of these cancers (11,12). Rab25 is also upregulated in prostate cancer and transitional-cell bladder cancer (11). Overexpression of other Rab proteins such as Rab5a and Rab7 in cancer has been reported (13,14).
A number of small molecule inhibitors of FTase have been developed and some of these are currently in clinical trials as anti-cancer therapeutics. See O'Regan, R. M., Khuri, F. R. (2004) Farnesyl transferase inhibitors: the next targeted therapies for breast cancer? Endocr. Relat. Cancer 11, 191-205; ClinicalTrials.gov: www.clinicaltrials.gov. Farnesyltransferase inhibitors (FTIs) exhibit clinical activities with leukemia, multiple myeloma, glioblastoma and advanced breast cancer. Ras proteins are farnesylated and that farnesylation and membrane association of the Ras proteins is critical for their ability to transform cells. Preclinical studies using mouse model systems driven by activated H-ras revealed dramatic ability of FTIs to inhibit tumor growth. However, subsequent studies demonstrated that FTIs are incapable of inhibiting prenylation of K-ras4B and N-ras, as these proteins are alternatively modified by geranylgeranyltransferase type I in the presence of FTIs. Thus, the effects of FTIs are speculated to be due to the inhibition of other farnesylated proteins such as Rheb, CENP-E, F, RhoB.
Compared with FTIs, development of geranylgeranyltransferase type I inhibitors (GGTIs) has lagged behind. Only a handful of compounds has been identified and most of these are derived from the CaaL peptide. See (a) Farnesyltransferase inhibitors in cancer therapy (eds. Sebti, S. M. and Hamilton, A. D.) Humana Press. (b) Oualid, F. E., van den Elst, H., Leroy, I. M., Pieterman, E., Cohen, L. H., Burm, B. E. A., Overkleeft, H. S., van der Marel, G. A., Overhand, M. (2005) A Combinatorial Approach toward the Generation of Ambiphilic Peptide-Based Inhibitors of Protein:Geranylgeranyl Transferase-I, J. Comb. Chem. 7, 703-713. (c) Carrico, D., Blaskovich, M. A., Bucher, C. J., Sebti, S. M., Hamilton, A. D. (2005) Design, synthesis, and evaluation of potent and selective benzoyleneurea-based inhibitors of protein geranylgeranyltransferase-I, Bioorg. Med. Chem. 13, 677-688. (d) Peterson, Y. K., Kelly, P., Weinbaum, C. A., Casey, P. J. (2006) A Novel Protein Geranylgeranyltransferase-I Inhibitor with High Potency, Selectivity and Cellular Activity, J. Biol. Chem. 
Chemical genomics intends to identify small molecule inhibitors of medically relevant targets in a systematic manner. While there are more than 100,000 proteins in the human body and approximately 10% of these are involved in human disease, only 500 have been exploited as drug targets. Thus, a vast amount of novel targets are present and identification of small molecule inhibitors of these unknown targets is of paramount importance. To maximize the chance of identifying small molecule compounds against these targets, a variety of libraries that have diverse structural motifs are needed. This is the goal of diversity-oriented synthesis (DOS). In contrast to combinatorial chemistry that aims to create a library of compounds derived from one scaffold, DOS aims to generate an array of compounds with different three-dimensional structures. See (a) Richter, H.; Walk, T.; Höltzel, A.; Jung, G. “Polymer Bound 3-Hydroxy-2-methylidenepropionic Acids. A Template for Multiple Core Structure Libraries” J. Org. Chem. 1999, 64, 1362-1365. (b) Purandare, A. V.; Gao, A.; Poss, M. A. “Solid-phase synthesis of ‘diverse’ heterocycles” Tetrahedron Lett. 2002, 43, 3903-3906. (c) Huang, X.; Liu, Z. “Solid-Phase synthesis of 4(1H)-Quinolone and Pyrimidine Derivatives Based on a New Scaffold—Polymer-Bound Cyclic Malonic Acid Ester” J. Org. Chem. 2002, 67, 6731-6737. (d) Couladouros E. A.; Strongilos, A. T. “Generation of Libraries of Pharmacophoric Structures with Increased Complexity and Diversity by Employing Polymorphic Scaffolds” Angew. Chem., Int. Ed. 2002, 41, 3677-3680. (e) Bertozzi, F.; Gundersen, B. V.; Gustafsson, M.; Olsson, R. “A Combinatorial Scaffold Approach Based upon a Multicomponent Reaction” Org. Lett. 2003, 5, 1551-1554. (f) Taylor, S. J.; Taylor, A. M.; Schreiber, S. L. “Synthetic Strategy toward Skeletal Diversity via Solid-Supported, Otherwise Unstable Reactive Intermediates” Angew. Chem., Int. Ed. 2004, 43, 1681-1685.
Diversity-oriented synthesis of a chemical compound library provides a powerful means to identify small molecule inhibitors against medically relevant targets. Initial screens of a pilot library followed by diversification using solid phase synthesis can yield potent inhibitors of enzymes in a relatively short period of time.
Since late 1990, a number of attempts to carry out diversity-oriented synthesis have been reported. One example is the use of squaric acid as a multireactive core molecule to generate a library consisting of different core structures. See Tempest, P. A. and Armstrong, R. W. (1997) Cyclobutenedione derivatives on solid support: Toward multiple core structure libraries. J. Am. Chem. Soc. 119, 7607-7608. However, very few of these examples were used in actual library synthesis. See (a) Ding, S.; Gray, N. S.; Wu, X.; Ding, Q.; Schultz, P. G. “A Combinatorial Scaffold Approach toward Kinase-Directed Heterocycle Libraries” J. Am. Chem. Soc. 2002, 124, 1594-1596. (b) Kwon, O.; Park, S. B.; Schreiber, S. L. “Skeletal Diversity via a Branched Pathway: Efficient Synthesis of 29,400 Discrete, Polycyclic Compounds and Their Arraying into Stock Solutions” J. Am. Chem. Soc. 2002, 124, 13402-13404. (c) Burke, M. D.; Berger, E. M.; Schreiber, S. L. “Generating Diverse Skeletons of Small Molecules Combinatorially” Science 2003, 302, 613-618. Due to drastically different reaction conditions, the yields greatly varied from one reaction to another, which discouraged adaptation of the methodology to a combinatorial library synthesis in solid-phase.
There thus exists a need for the identification of a multireactive core molecule for which more consistent reaction conditions can be used and with which more consistent yield is obtained in diversity oriented synthesis. There exists a need for novel GGTI and RabGGTase inhibiting compounds.